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Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Striatin (STRN) is a multivalent protein holding great therapeutic potentials in view of its interaction with dynamic partners implicated in apoptosis. Although striatin-3 and striatin-4, that share high structural similarities with STRN, have been linked to apoptosis, the dynamics of STRN in apoptotic cells remain unclear. Herein, we report that the amount of STRN (110 kDa) is reduced in apoptotic cells, in response to various chemotherapeutic agents, thereby yielding a major polypeptide fragment at ~65 kDa, and three minor products at lower molecular weights. While STRN siRNA reduced the 65 kDa derivative fragment, the overexpression of a Myc-tagged STRN precipitated a novel fragment that was detected slightly higher than 65 kDa (due to the Myc-DDK tag on the cleaved fragment), confirming the cleavage of STRN during apoptosis. Interestingly, STRN cleavage was abrogated by the general caspase inhibitor Z-VAD.fmk. Cell fractionation revealed that the STRN pool, mainly distributed in the non-cytosolic fragment of naïve cells, translocates to the cytosol where it is proteolytically cleaved during apoptosis. Interestingly, the ectopic expression of caspase 3 in MCF-7 cells (deprived of caspase 3) induced STRN cleavage under apoptotic conditions. Inhibition of caspase 3 (Ac-DEVD-CHO) conferred a dose-dependent protection against the proteolytic cleavage of STRN. Collectively, our data provide cogent proofs that STRN translocates to the cytosol where it undergoes proteolytic cleavage in a caspase 3-dependent manner during apoptosis. Thus, this study projects the cleavage of STRN as a novel marker for apoptosis to serve pharmacological strategies targeting this particular form of cell death., Cell biology; Cell culture; Cell death; Cytoskeleton; Membrane; Striatin, apoptosis, caspase 3, and

Congenital heart diseases (CHDs) are complex traits that manifest in diverse clinical phenotypes such as the Tetralogy of Fallot (TOF), valvular and ventricular/atrial septal defects. Genetic mechanisms of CHDs have remained largely unclear to date. Copy number variations (CNVs) have been implicated in many complex diseases but their impact has not been examined extensively in various forms of CHD lesions. We report in this study, to the best of our knowledge, the largest cohort of Saudi Arab CHD patients to date who were evaluated using genome-wide CNV analysis. In a sample of 134 Saudi Arab patients with CHD, 66 exhibited pathogenic or likely pathogenic CNVs. Notably, 21 copy number gains and 11 copy number losses were detected that encompassed 141 genes and 146 genes, respectively. The most frequent gains were on 17q21.31, 8p11.21, and 22q11.23, whereas the losses were primarily localized to 16p11.2. Interestingly, all lesions have had gains at 17q21.31. Septal defects had also gains at 8p11.21 and 22q11.23, valvular lesions at 8p11.21, 22q11.23, and 2q13, and TOF at 16p11.2. Functional and network analyses demonstrated that cardiovascular and nervous system development and function as well as cell death/survival were most significantly associated with CNVs, thus highlighting the potentially important genes likely to be involved in CHD, including NPHP1, PLCB1, KANSL1, and NR3C1. In conclusion, this genome-wide analysis identifies a high frequency of CNVs mostly in patients with septal defects, primarily influencing cardiovascular developmental and functional pathways, thereby offering a deeper insight into the complex networks involved in CHD pathogenesis.

Publisher's version (útgefin grein)., In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44–66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding., This paper is the work of the ROHgen consortium. We thank the Sigma T2D Consortium, whose members are detailed in Supplementary Note 3. We thank the UK Biobank Resource, approved under application 19655; we acknowledge funding from the UK Medical Research Council Human Genetics Unit and MRC Doctoral Training Programme in Precision Medicine. We also thank Neil Robertson, Wellcome Trust Centre for Human Genetics, Oxford, for use of his author details management software, Authorial. Finally, we thank all the participants, researchers and funders of ROHgen cohorts. Cohort-specific acknowledgements are in Supplementary Data 2; personal acknowledgements and disclosures are in Supplementary Note 2. We thank Rachel Edwards for administrative assistance.

The genetic susceptibility to acquiring low high density lipoprotein-cholesterol (LHDLC) levels is not completely elucidated yet. In this study, we performed a common variant association study for harboring this trait in ethnic Arabs. We employed the Affymetrix high-density Axiom Genome-Wide ASI Array (Asian population) providing a coverage of 598,000 single nucleotide variations (SNPs) to genotype 5495 individuals in a two-phase study involving discovery and validation sets of experiments. The rs1800775 [1.31 (1.22-1.42); p = 3.41E-12] in the CETP gene and rs359027 [1.26 (1.16-1.36); p = 2.55E-08] in the LMCD1 gene were significantly associated with LHDLC levels. Furthermore, rs3104435 [1.26 (1.15-1.38); p = 1.19E-06] at the MATN1 locus, rs9835344 [1.16 (1.08-1.26); p = 8.75E-06] in the CNTN6 gene, rs1559997 [1.3 (1.14-1.47); p = 9.48E-06] in the SDS gene and rs1670273 [1.2 (1.1-1.31); p = 4.81E-06] in the DMN/SYNM gene exhibited suggestive association with the disorder. Seven other variants including rs1147169 in the PLCL1 gene, rs10248618 in the DNAH11, rs476155 in the GLIS3, rs7024300 in the ABCA1, intergenic rs10836699, rs11603691 in P2RX3 and rs750134 in CORO1C gene exhibited borderline protective properties. Validation and joint meta-analysis resulted in rs1800775, rs3104435 and rs359027 retaining their predisposing properties, while rs10836699 and rs11603691 showed protective properties. Our data show several predisposing variants across the genome for LHDLC levels in ethnic Arabs.

Over 120 Type 2 diabetes (T2D) loci have been identified from genome-wide association studies (GWAS), mainly from Caucasian populations. Very limited knowledge is available on the Saudi Arabian population. In this study, 122 previously reported T2D-related variants from 84 loci were examined in a Saudi Arabian cohort of 1,578 individuals (659 T2D cases and 919 controls). Eleven single nucleotide polymorphisms (SNPs) corresponding to nine independent loci had a P value −4 ( = 0.05/122) were applied, none of the SNPs would have reached the significance level. Nine of the SNPs with a P value

Genome-wide association studies (GWASs) have yielded a wealth of information furnishing support for the variability in genetic predisposition to disease. However, the actual impact of such findings on any particular ethnic population needs to be validated through replication studies. In the present study, we verified recent findings of a GWAS demonstrating a strong association for the cyclin-dependent kinase 4 inhibitor B (CDKN2B) genomic region with coronary artery disease (CAD)/myocardial infarction (MI) in ethnic Saudi Arabs.We genotyped 8 CDKN2B SNPs for cardiovascular risk in 4650 Saudi Arabs, comprising 2429 CAD cases (1860 males; 569 female) and 2221 controls (1189 male; 1032 female) by Taqman assay.Four SNPs, rs4977574_A [0.56(0.50-0.63); p 0.0001], rs10757274_A [0.87(0.77-0.97); p = 0.014], rs10738607_A [0.89(0.80-1.00); p = 0.043] and rs1333045_T [0.54(0.48-0.61); p 0.0001] residing on the CDKN2B gene were significantly associated with CAD following multivariate adjustments for MI, HTN and DM, while four others were weakly associated with the disease. Likewise, three SNPs, rs1412829_G [0.84(0.72-0.97); p = 0.019], rs564398_C [0.81(0.70-0.94); p = 0.006], rs4977756_G [0.87(0.76-0.99); p = 0.036] were significantly associated with MI after multivariate adjustments for CAD, HTN and DM, while the other five displayed borderline associations.Our findings strongly support the notion of a critical role for the CDKN2B gene locus as a cardiovascular risk in ethnic Arabs. The study also demonstrates the importance of replication studies in ascertaining the role of a genomic sequence in disease.

The sodium iodide symporter ( NIS ) (solute carrier family 5; SLC5A), mediates the active transport of iodine anion (I − ) into thyroid follicular cells to facilitate thyroid hormone biosynthesis. Considering its fundamental role in thyroid function, our objective in this study is to explore its potential involvement in the pathogenesis of differentiated thyroid cancer (DTC). Following a preliminary sequencing of the gene in a representative sample of the general population, five variants, (1) rs45602038, (2) rs4808708, (3) rs4808709, (4) rs7250346 and (5) rs12327843, were selected for a larger population-based association study consisting of 507 cases and 597 controls, of which only the rs45602038_TT [Odds ratio (95% confidence interval) = 1.90 (1.26–2.88); p = 0.002] was associated with disease following adjustment for other confounders using the multivariate analysis. Furthermore, a 5-mer haplotype CGAGT constructed from the five studied SNPs conferred a significant risk ( χ 2 = 10.98; p = 0.0009) for DTC. This association trickled down through shorter derivatives, with the 4-mer haplotype CGAG ( χ 2 = 13.25; p = 0.0003) displaying the most significant association and the 3-mer GAG ( χ 2 = 11.80; p = 0.0006) being equally strongly linked to the disease. Comparison of the flanking derivatives of the primary 5-mer haplotype also indicated that the 3-mer CGA ( χ 2 = 4.04; p = 0.045) constructed from SNP block 1–3 was a lot weaker than that of the AGT ( χ 2 = 6.73; p = 0.0095) constructed from the blocks 3–5 from the other end of the gene. Put together, these data implicate the three nucleotide changes at the rs4808708, rs4808709 and rs7250346 loci (blocks 2–4) as the core for this relationship.

Objectives The drug-metabolizing enzymes and transporters (DMET) Plus microarray and x-Tag assays have recently been developed for genotyping individuals in personalized medicine. Furthermore, the cytochrome 450-2C19 (CYP2C19) is a key metabolic enzyme encoded by a polymorphic gene commonly associated with diminished metabolism and variable clinical responses to several drugs in an ethnicity-dependent fashion. Therefore, validation of these clinical procedures as well as knowledge of the ethnic-specific incidences of these gene variants is prerequisite for determining their clinical relevance in any given population. Methods We determined the distribution of familiar CYP2C19 variants by the DMET Plus chip in 600 candidates and replicated the findings by the Affymetrix Axiom Genome-Wide Asian Structure Identification Array in 5413 individuals, all Saudis of ethic Arab origin. We then tested the robustness of employing the Luminex xMAP system clinically by comparing the results of genotyping 500 Saudi individuals visiting the Blood Bank of our institution with the findings of the two platforms. Key findings The DMET Plus genotyping revealed that eight of the CYP2C19 variants showed some changes. Thereby, the CYP2C19*17 exhibited the highest minor allele frequency (MAF) of 0.256, followed by the CYP2C19_801 (frequency = 0.055). Six other variants, including the CYP2C19*3, showed MAF in the range of 0.001–0.002. We replicated the frequencies of the CYP2C19*17 and CYP2C19*3, and additionally established that of the CYP2C19*2 (0.099) using the Axiom platform. The xTag genotyping also indicated that 0.834 of the 500 Saudi individuals were extensive metabolizers (*1/*1), 0.158 carried the *1/*2 genotype, 0.01% carried *2/*2 (poor metabolizers) and one each (0.2%) harboured the *1/*8, *2/*3 (intermediate metabolizers) and *8/*8 (poor metabolizers) genotypes. Conclusions The results showed reproducible genotyping of the CYP2C19 variants in the Saudi Arab population using two Affymetrix platforms and phenotyping using the Luminex xTag assay. The prevalence of two clinically relevant genotypes (CYP2C19*2 and CYP2C19*3) were similar to other ethnic groups, while that of the CYP2C19*17 was comparably higher.

Impaired cardiomyocyte contraction rate is detrimental to cardiac function and often lethal. Despite advancements in the field, there is a paucity of information regarding the coordination of molecules implicated in regulating the heart rate. Striatin (STRN) is a dynamic protein with binding domains to calmodulin (CaM) and caveolin (Cav), both of which are regulators of myocardial function. However, its role in cardiomyocyte contraction is not yet determined. Herein, we show that STRN is expressed in cardiomyocytes and is more abundant in atrial myocardium than in ventricles. Cardiac expression of STRN (protein and mRNA) was developmentally regulated with the highest expression being at neonatal stage (day one) and the lowest in adult rats (13 weeks). CaM pulldown assay indicated that the interaction of cardiac STRN with CaM and caveolin-3 (Cav-3) was calcium sensitive. Interestingly, the overexpression of STRN induced an increase (∼2-fold) in the rate of the spontaneous contraction of cultured cardiomyocytes, while the knockdown of STRN reduced their contraction rate (∼40%). The expression level of STRN was inversely proportional to the interaction of Cav-3 with the CaM/STRN complex. Collectively, our data delineate a novel role for STRN in regulating cardiomyocyte spontaneous contraction rate and the dynamics of the STRN/Cav-3/CaM complex.

Background Congenital long QT syndrome (LQTS) is an inherited, potentially fatal arrhythmogenic disorder. At least 16 genes have been implicated in LQTS; the yield of genetic analysis of 3 genes ( KCNQ1 , KCNH2 , and SCN5A ) is about 70%, with KCNQ1 mutations accounting for ∼50% of positive cases. LQTS is mostly inherited in an autosomal dominant pattern. Systemic analysis of LQTS has not been previously conducted in a population with a high degree of consanguinity. Objectives To describe the clinical and molecular profiles of LQTS in the highly consanguineous Saudi population. Methods Fifty-six Saudi families with LQTS were consecutively recruited and evaluated. Sequencing of KCNQ1 , KCNH2 , and SCN5A genes was conducted on all probands, followed by screening of family relatives. Results Genetic analysis was positive in 32 (57.2%) families, with mutations in KCNQ1 identified in 28 families (50%). Surprisingly, 17 (53.1%) probands were segregating homozygous mutations. Family screening identified 123 individuals with mutations; 89 (72.4%) were heterozygous, 23 (18.7%) were homozygous, and 11 (8.9%) were compound heterozygous. Compared to heterozygous, the phenotype was more severe in homozygous individuals, with cardiac symptoms in 78.3% (vs 12.4%), family history of sudden death in 64.7% (vs 44.4%), and prolonged QT interval in 100% (vs 43.8%). Congenital deafness was found in 11 (47.8%) homozygous probands. Conclusion Our study provides insight into the clinical and molecular profiles of LQTS in a consanguineous population. It underscores the importance of preemptive management in homozygous patients with LQTS and the value of clinical and molecular screening of at-risk relatives.

This publication is a correction of the thirty-third author’s name. The URL of the article is: https://www.um.edu.mt/library/oar/handle/123456789/25240, peer-reviewed

The GATA2 is a multi-catalytic transcription factor believed to play an important role in regulating inflammatory processes, largely contributory to cardiovascular-related events. However, its role in coronary artery disease (CAD) risk traits remains poorly understood. In a preliminary study using Affymetrix 250K, we established a link on chromosome (chr) 3, which harbors the GATA2 gene, to early onset of CAD in two families with heterozygous familial hyperlipidemia (HFH), suggesting a role for the gene in metabolic-related CAD in the general population. We then sequenced the gene in the families and an additional 200 individuals in the general population, followed by an association study for 8 SNPs on CAD metabolic risk traits in a total of 4557 individuals (2386 CAD cases versus 2171 angiographed controls) by the Applied Biosystems real-time PCR system. The rs1573949_C [1.15(1.00–1.32); p = 0.049] was associated with MI, rs7431368_AA [5.2(1.05–26.60); p = 0.43] conferred risk for harboring low high density lipoprotein, and obesity was linked to rs10934857_AA [5.69(1.04–30.98); p = 0.045] following Bonferroni corrections and multivariate adjustments for confounders. Furthermore, a haplotype CCCGGGTC (χ2 = 4.23; p = 0.04) constructed from the eight studied SNPs and its 6-mer derivative CGGGTC (χ2 = 5.05; p = 0.025) were associated with CAD. Obesity was associated with the 6-mer CATAAA (χ2 = 3.66; p = 0.049), and hypercholesterolemia was linked to the 8-mer CCTGGACC (χ2 = 6.02; p = 0.014), but most significantly so with its 5-mer derivative, CTGGA (χ2 = 6.75; p = 0.009). On the other hand, high low density lipoprotein was linked to TGG (χ2 = 4.48; p = 0.034). Our study points to an association of GATA2 at both SNP and haplotype levels with important metabolic risk traits for atherosclerosis.

Congenital long QT syndrome (LQTS) is an inherited potentially fatal arrhythmogenic disorder that is characterized by prolonged corrected QT (QTc) interval. Mutations in three genes (KCNQ1, KCNH2, and SCN5A) account for the majority of the cases. However, 10 other genes are now known to be implicated in LQTS. In this work, we describe the clinical and molecular analysis in a large Saudi family with LQTS. Screening KCNQ1, KCNH2, and SCN5A genes in the proband, who presented with syncope, led to the identification of a heterozygous mutation (p.H258P) in KCNQ1. An extended clinical and genetic screening of the family identified 11 other members who were carriers for this mutation. All identified carriers had prolonged QTc intervals; yet, only two were symptomatic. Screening the family members for three LQTS modifiers (rs4657139 and rs16847548 in NOS1AP and KCNE1-D85N) did not reveal a correlation with symptoms or QTc intervals. The electrocardiographic and molecular analysis stratified seven carriers at high risk of a cardiac event as they had a QTc of ≥500 ms and were carriers of a KCNQ1 mutation. Our work illustrates the importance of extended family screening in LQTS to identify silent carriers and hence adopt the most appropriate therapeutic and preventive intervention.

AIMS:The disease pathways leading to idiopathic dilated cardiomyopathy (DCM) are still elusive. The present study investigated integrated global transcriptional and translational changes in human DCM for disease biomarker discovery. METHODS:We used identical myocardial tissues from five DCM hearts compared to five non-failing (NF) donor hearts for both transcriptome profiling using the ABI high-density oligonucleotide microarrays and proteome expression with One-Dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on the Synapt G2 system. RESULTS:We identified 1262 differentially expressed genes (DEGs) and 269 proteins (DEPs) between DCM cases and healthy controls. Among the most significantly upregulated (>5-fold) proteins were GRK5, APOA2, IGHG3, ANXA6, HSP90AA1, and ATP5C1 (p< 0.01). On the other hand, the most significantly downregulated proteins were GSTM5, COX17, CAV1 and ANXA3. At least ten entities were concomitantly upregulated on the two analysis platforms: GOT1, ALDH4A1, PDHB, BDH1, SLC2A11, HSP90AA1, HSP90AB1, H2AFV, HSPA5 and NDUFV1. Gene ontology analyses of DEGs and DEPs revealed significant overlap with enrichment of genes/proteins related to metabolic process, biosynthetic process, cellular component organization, oxidative phosphorylation, alterations in glycolysis and ATP synthesis, Alzheimer's disease, chemokine-mediated inflammation and cytokine signalling pathways. CONCLUSION:The concomitant use of transcriptome and proteome expression to evaluate global changes in DCM has led to the identification of sixteen commonly altered entities as well as novel genes, proteins and pathways whose cardiac functions have yet to be deciphered. This data should contribute towards better management of the disease.

Hypertriglyceridemia (hTG) is a lipid disorder, resulting from an elevation in triglyceride levels, with a strong genetic component. It constitutes a significant risk factor for coronary artery disease (CAD), a leading cause of death worldwide. In this study, we performed a common variant association study for hTG in ethnic Saudi Arabs. We genotyped 5501 individuals in a two-phase experiment using Affymetrix Axiom® Genome-Wide CEU 1 Array (Affymetrix, Santa Cruz, CA) that contains a total of 587,352 single nucleotide polymorphisms (SNPs). The lead variant was the rs1558861 [1.99 (1.73-2.30); p = 7.37 × 10-22 ], residing on chromosome (chr) 11 at the apolipoprotein A-I/A-5 (APOA1/APOA5) locus. The rs780094 [1.34 (1.21-1.49); p = 8.57 × 10-8 ] on chr 2 at the glucokinase regulatory protein (GCKR) locus was similarly significantly associated, while the rs10911205 [1.29 (1.16-1.44); p = 3.52 × 10-6 ] on chr1 at the laminin subunit gamma-1 (LAMC1) locus showed suggestive association with disease. Furthermore, the rs17145738 [0.68 (0.60-0.77); p = 6.69 × 10-9 ] on chr7 at the carbohydrate-responsive element-binding protein-encoding (MLXIPL) gene locus displayed significant protective characteristics, while another variant rs6982502 [0.76 (0.68-0.84); p = 5.31 × 10-7 ] on chr8 showed similar but weaker properties. These findings were replicated in 317 cases vs 1415 controls from the same ethnic Arab population. Our study identified several variants across the human genome that are associated with hTG in ethnic Arabs.

Clinical characteristics of the studied family. (DOCX 82Â kb)

Coverage and sequencing depth of the exome data. (DOCX 17Â kb)

Background Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes, mostly encoding sarcomeric proteins and proteins of the cytoskeleton, have been implicated in familial DCM to date. Yet, the majority of variants causing DCM remain to be identified. The goal of the study is to identify novel mutations causing familial dilated cardiomyopathy. Results We identify FBXO32 (ATROGIN 1), a member of the F-Box protein family, as a novel DCM-causing locus. The missense mutation affects a highly conserved amino acid and is predicted to severely impair binding to SCF proteins. This is validated by co-immunoprecipitation experiments from cells expressing the mutant protein and from human heart tissue from two of the affected patients. We also demonstrate that the hearts of the patients with the FBXO32 mutation show accumulation of selected proteins regulating autophagy. Conclusion Our results indicate that abnormal SCF activity with subsequent impairment of the autophagic flux due to a novel FBXO32 mutation is implicated in the pathogenesis of DCM. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0861-4) contains supplementary material, which is available to authorized users.

By virtue of the functional role of the mitochondrion in energy and reactive oxygen species production, mutations in mitochondrial DNA (mtDNA) are potential candidates for cardiovascular-related disorders. Further, the mtDNA is extremely polymorphic and several diagnostic single-nucleotide polymorphisms have been used to assort sequences into haplogroups with defined continental and regional ranges. However, the relevance of these haplogroups and mutations with respect to coronary artery disease (CAD) susceptibility remains unclear. In this study, we evaluated the role of the 16189TC variants and mtDNA haplogroups as predisposing factors for CAD in 669 Saudi patients with angiographically established disease compared with 258 disease-free controls. The 16189TC was associated with CAD (1.524 [1.076-2.159]; p = 0.017). However, this association was influenced by age as well as the presence of myocardial infarction and hypertension. Among the haplogroups, only the N1c showed a borderline protective relationship (p = 0.074) with CAD as an independent risk factor. This association turned significant in the total sample (0.176 [0.042-0.736]; p = 0.017) and in the50-year age group (0.075 [0.008-0.743]; p = 0.027), when included as a possible confounding factor. Our results suggested that the impact of mtDNA polymorphism on CAD manifestation is influenced by important confounders, particularly the presence of myocardial infarction, hypertension, and age.

Summary The role of the KIAA0391 and PSMA6 genes in predisposing individuals to disease is still not fully understood. We evaluated by molecular beacon-based genotyping assays the roles of five single nucleotide polymorphisms (SNPs) in the chromosomal region 14q13.2 harbouring the KIAA0391 and PSMA6 gene cluster in coronary artery disease (CAD) in the Saudi population. Two of the studied SNPs rs8008319 (denoted as 1) and rs7157492 (2), reside in the KIAA0391 locus, two others rs1048990 (3) and rs12878391 (4) are components of the PSMA6, while rs4981283 (5) resides downstream of both genes. In a study involving 1071 patients and 929 controls, none of the studied SNPs showed significant association with CAD. In contrast, two haplotypes consisting of 1A-2G-3C-4A-5A [O.R.(95% C.I.) = 1.49(0.95–2.35); p = 0.022] and 1A-2G-3G-4A-5A [2.24(0.84–5.98); p = 0.031] conferred risk for both CAD and myocardial infarction (MI) in a five-SNP locus model, while another comprising 1A-2G-3C-4A-5G [2.24(0.84–5.98); p = 0.079] showed a borderline association. One haplotype consisting of 1T-2G-3C-4G-5A [0.79(0.59–1.05); p = 0.015] exhibited protective properties and another, 1T-2G-3C-4A-5G [0.20(0.03–139); p = 0.073], showed a similar but weaker trend. Our study identified haplotypes in the chromosomal region encompassing the KIAA0391 and PSMA6 genes as a possible genetic link between CAD and MI. These results also suggest that haplotypes may be more informative than individual SNPs in identifying risk factors for disease.

We employed ABI high-density oligonucleotide microarrays containing 31,700 sixty-mer probes (representing 27,868 annotated human genes) to determine differential gene expression in idiopathic dilated cardiomyopathy (DCM). We identified 626 up-regulated and 636 down-regulated genes in DCM compared to controls. Most significant changes occurred in the tricarboxylic acid cycle, angiogenesis, and apoptotic signaling pathways, among which 32 apoptosis- and 13 MAPK activity-related genes were altered. Inorganic cation transporter, catalytic activities, energy metabolism and electron transport-related processes were among the most critically influenced pathways. Among the up-regulated genes were HTRA1 (6.9-fold), PDCD8(AIFM1) (5.2) and PRDX2 (4.4) and the down-regulated genes were NR4A2 (4.8), MX1 (4.3), LGALS9 (4), IFNA13 (4), UNC5D (3.6) and HDAC2 (3) (p

Background Heterogeneous familial hypercholesterolemia (HFH) partly underlies polymorphic changes in the low-density lipoprotein receptor ( LDLR ), apolipoprotein B ( APOB ) and protein convertase subtilisin/kexin type 9 ( PCSK9 ), exhibiting intra-ethnical variations in its clinical features. Methods We employed the Affymetrix whole genome scan 250 sty1 array to characterize possible genomic linkage to heterozygous familial hypercholesterolemia (HFH) and sequencing techniques to identify related mutations in the above three genes in a Saudi family of 11 individuals harbouring clinical features of FH. The propositus had early onset of coronary artery disease (CAD) and very significantly elevated cholesterol (Chol) level of 10.1 mmol/L and LDL-cholesterol (LDL-C) of 7.9 mmol/L as well as low HDL-C level of 0.51 mmol/L, while 4 siblings were affected with HFH. Results Whole genome scan for the autosomal dominant model showed high homology for the affected individuals in several regions including chromosomes (chr) 1 and 2 which harbour PCSK9 and APOB , respectively. Subsequent sequencing of the coding regions of these two and LDLR identified 11 single nucleotide polymorphisms (SNPs) in the LDLR , 8 in the APOB and 6 in the PCSK9 genes. The propositus uniquely carried the homozygous mutant genotypes (haplotype) for all 11 LDLR SNPs, in direct contrast to the only normolipidemic sibling and a control who carried the homozygous wild type genotypes at these loci. Another set of 7 SNPs in the APOB also isolated with FH. Interestingly, all family members were heterozygous for all except the rs2228671 C > T of this gene, for which the mother shared the C/C genotype with the propositus, two other affected off-springs and a control, all of whom exhibited low HDL-C levels. A confirmation experiment involving 70 individuals harbouring low HDL-C revealed 74.3% of them as C/C carriers. Conclusions Our study identified a haplotype in the LDLR as a marker for early onset of CAD, and rs2228671 C > T in the LDLR in association with a reduction in HDL-C concentrations in FH. The results also substantiate the notion of genetic heterogeneity in HFH, underlining the essence of recognizing ethnic-specific gene variability as a potential basis for appropriate management of FH.

Cardiomyocyte apoptosis is implicated in the pathogenesis of heart failure and mitochondria play an important role in this mode of cell death. In this study, activity of the Bcl-2 family of mitochondrial apoptotic proteins and their regulator (p53) were assessed during the transition to heart failure.Ten adult male sheep were banded with a variable aortic constriction device. This was progressively inflated to increase left ventricular (LV) afterload. The sheep were monitored echocardiographically, measuring LV Mass Index (LVMI), diastolic LV Internal Diameter (LVIDd) and Fractional Shortening (FS). Serial LV endomyocardial biopsies were obtained, to measure expression of p53 and Bcl-2 family proteins by Western blotting.Over the first 3-4 weeks, the sheep developed hypertrophy (LVMI 79.5+/-4.6 vs. 44.0+/-3.0 g/m2, p0.01), followed by gradual LV dilatation (LVIDd 4.23+/-0.08 vs. 3.39+/-0.07 cm, p0.01). Ventricular function remained stable until 7-8 weeks post banding, when there was significant deterioration (FS 18.3+/-2.4 vs. 46.9+/-2.6%, p0.01), associated with clinical heart failure. Upregulation of the Bax/Bcl-2 ratio, associated with increased levels of p53, was demonstrated in each of the echocardiographically defined stages (LV hypertrophy, LV dilatation and LV failure). In parallel, significantly higher levels of anti-apoptotic protein (Bcl-xL) was associated with LV dilatation and failure.Upregulation of Bax/Bcl-2 ratio occurs during the transition to heart failure and in particular with the initial hypertrophic response. Increase in expression of the anti-apoptotic protein Bcl-xL suggests possible concomitant compensatory mechanisms being activated during the transition to heart failure.

Advances in depression research have targeted inflammation and oxidative stress to develop novel types of treatment. The JAK/STAT signaling pathway plays pivotal roles in immune and inflammatory responses. The present study was designed to investigate the effects of N-acetylcysteine, a putative precursor of the antioxidant glutathione, in an animal model of depression, with an emphasis on the JAK/STAT signaling pathway. Fluoxetine, a classical antidepressant drug was also under investigation. Male Wistar rats were subjected to forced swimming test and given N-acetylcysteine and fluoxetine immediately after the pre-test session, 5 h later and 1 h before the test session of the forced swimming test. N-acetylcysteine decreased immobility time ( P

A disease phenotype generally reflects various pathobiological processes that interact in a complex network. The highly interconnected nature of the human protein interaction network (interactome) indicates that, at the molecular level, it is difficult to consider diseases as being independent of one another. Recently, genome-wide molecular measurements, data mining and bioinformatics approaches have provided the means to explore human diseases from a molecular basis. The exploration of diseases and a system of disease relationships based on the integration of genome-wide molecular data with the human interactome could offer a powerful perspective for understanding the molecular architecture of diseases. Recently, subnetwork markers have proven to be more robust and reliable than individual biomarker genes selected based on gene expression profiles alone, and achieve higher accuracy in disease classification. We have applied one of these methodologies to idiopathic dilated cardiomyopathy (IDCM) data that we have generated using a microarray and identified significant subnetworks associated with the disease. In this paper, we review the recent endeavours in this direction, and summarize the existing methodologies and computational tools for network-based analysis of complex diseases and molecular relationships among apparently different disorders and human disease network. We also discuss the future research trends and topics of this promising field.

Background Multiple loci have been identified for coronary artery disease (CAD) by genome-wide association studies (GWAS), but no such studies on CAD incidence has been reported yet for any Middle Eastern population. Methods In this study, we performed a GWAS for CAD and myocardial infarction (MI) incidence in 5668 Saudis of Arab descent using the Affymetrix Axiom Genotyping platform. Results We describe SNPs at 16 loci that showed significant (P −8 ) or suggestive GWAS association (P −5 ) with CAD or MI, in the ethnic Saudi Arab population. Among the four variants reaching GWAS significance in the present study, the rs10738607_G [0.78(0.71–0.85); p = 2.17E-08] in CDNK2A/B gene was associated with CAD. Two other SNPs on the same gene, rs10757274_G [0.79(0.73–0.86); p = 2.98E-08] and rs1333045_C [0.79(0.73–0.86); p = 1.15E-08] as well as the rs9982601_T [1.38(1.23–1.55); p = 3.49E-08] on KCNE2 were associated with MI. These variants have been previously described in other populations. Several SNPs, including the rs7421388 ( PLCL1) and rs12541758 ( TRPA1) displaying a suggestive GWAS association (P −5 ) with CAD as well as rs41411047 ( RNF13 ), rs32793 ( PDZD2 ) and rs4739066 ( YTHDF3 ), similarly showing weak association with MI, were confirmed in an independent dataset. Furthermore, our estimation of heritability of CAD and MI based on observed genome-wide sharing in unrelated Saudi Arabs was approximately 33% and 44%, respectively. Conclusions Our study has identified susceptibility variants for CAD/MI in ethnic Arabs. These findings provide further insights into pathways contributing to the susceptibility for CAD and will enable more comprehensive genetic studies of these diseases in Middle East populations.

Background. The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus Premier Pack has been designed to genotype 1936 gene variants thought to be essential for screening patients in personalized drug therapy. These variants include the cytochrome P450s (CYP450s), the key metabolizing enzymes, many other enzymes involved in phase I and phase II pharmacokinetic reactions, and signaling mediators associated with variability in clinical response to numerous drugs not only among individuals, but also between ethnic populations.Materials and Methods. We genotyped 600 Saudi individuals for 1936 variants on the DMET platform to evaluate their clinical potential in personalized medicine in ethnic Arabs.Results. Approximately 49% each of the 437 CYP450 variants, 56% of the 581 transporters, 56% of 419 transferases, 48% of the 104 dehydrogenases, and 58% of the remaining 390 variants were detected. Several variants, such as rs3740071, rs6193, rs258751, rs6199, rs11568421, and rs8187797, exhibited significantly either higher or lower minor allele frequencies (MAFs) than those in other ethnic groups.Discussion. The present study revealed some unique distribution trends for several variants in Arabs, which displayed partly inverse allelic prevalence compared to other ethnic populations. The results point therefore to the need to verify and ascertain the prevalence of a variant as a prerequisite for engaging it in clinical routine screening in personalized medicine in any given population.

The substitution of tryptophan (Trp) by arginine (Arg) at posi- tion 64 in the 3-adrenoceptor (3-AR) gene has been associated with obe- sity, diabetes mellitus, and coronary artery disease (CAD). We have investigated whether the Trp64Arg polymorphism is associated with the manifestation of CAD or one of its important risk factors, such as obesity, diabetes mellitus, elevated cholesterol and triglyceride levels, or hyperten- sion in the Arab population. All participating subjects were genotyped for this polymorphism using the polymerase chain reaction followed by enzy- matic digestion and sequencing. In the angiographed normal control subjects (n495), 90.3% were homozygous Trp/Trp, 9.5% were heterozygous Trp/ Arg, and 0.2% were homozygous for the Arg/Arg genotype, compared to 87%, 12.3%, and 0.7%, respectively, among angiographically confirmed CAD patients (n981). There was no statistical difference in the distribu- tion of genotypes or allele frequencies between the CAD and control groups. We carried out a stepwise logistic regression analysis to study the possible combined effect of the genotypes and other risk factors on CAD. All vari- ables were retained in the model, with p values of 0.014, 0.006, 0.005, 0.001, 0.045, 0.002, 0.001, and 0.016 for genotype, diabetes mellitus, sex, family history of CAD, obesity, myocardial infarction, smoking, and age, respectively. In conclusion, the Trp64Arg polymorphism of the 3-AR gene does not represent an independent risk factor for CAD in Arabs. How- ever, in the presence of other CAD risk factors, this polymorphism may be used as a predictor of CAD. The 3-adrenoceptor (3-AR) forms an essential component of the cardiovascu- lar sympathetico-adrenoceptor signaling systems. Human 3-AR is expressed

SUMMARY 1. Studies using animal experimental models have suggested that the β2-adrenoceptor is uncoupled in association with alterations in the expression of G-protein-coupled receptor kinases (GRK) 2/3 in heart failure. However, the functional expression of the components of this pathway in human disease has not been fully elucidated yet. In the present study, we evaluated the possibility that the regulation of β2-adrenoceptor signalling components in patients with left ventricular volume overload (VOL) depends on the severity of the overload. 2. We characterized the lymphocyte GRK 2–6, β-arrestins 1 and 2, β2-adrenoceptor expression at the mRNA and protein levels, as well as the activity of adenylyl cyclase, protein kinases (PK) A and PKC in patients with VOL using healthy blood donors as controls. 3. In the patient group, GRK2 mRNA was increased by 61% (P < 0.001), GRK3 was increased by 54% (P < 0.005), GRK5 was increased fivefold (P < 0.001) and the β-arrestin 2 mRNA was increased by 40% (P < 0.05). These increases were paralleled with a sixfold increase in GRK2, a twofold increase in GRK3 and a 1.3-fold increase in GRK5 protein levels. These changes were associated with a significant decrease in β2-adrenoceptor mRNA, the basal, catalytic and receptor-mediated activity of adenylyl cyclase and sensitization of the forskolin-stimulated activity towards augmented inhibition by guanylimidodiphosphate. In general, the increase in GRK2 and 5 mRNA exhibited a positive correlation with the gravity of the haemodynamic load, as determined by changes in left ventricular fractional shortening. 4. The results suggest that VOL induces an increase in the expression of lymphocyte β2-adrenoceptor-specific GRK and β-arrestin 2 in association with an attenuation in β2-adrenoceptor levels. It can be speculated that the cardiac circulatory system adapts itself to altered haemodynamic functional demands partly by altering β2-adrenoceptor signalling.

Although cardiac arrhythmias are still a major health threat for human, there is paucity of information on the molecular mechanisms involved in this pathology. Striatin (STRN) is a multivalent prot...

We examined the role of hepatic nuclear factor-1 alpha (HNF1a) gene polymorphism on coronary artery disease (CAD) traits in 4631 Saudi angiographed individuals (2419 CAD versus 2212 controls) using TaqMan assay on ABI Prism 7900HT sequence detection system. Following adjustment for confounders, the rs2259820_CC (1.19 (1.01–1.42);P=0.041), rs2464196_TT (1.19 (1.00–1.40);P=0.045), and rs2259816_T (1.13 (1.01–1.26);P=0.031) were associated with MI. The rs2259820_T (1.14 (1.03–1.26);P=0.011) and rs2464196_C (1.12 (1.02–1.24);P=0.024) were associated with type 2 diabetes mellitus (T2DM), while the rs2393791_T (1.14 (1.01–1.28);P=0.032), rs7310409_G (1.16 (1.03–1.30);P=0.013), and rs2464196_AG+GG (1.25 (1.05–1.49);P=0.012) were implicated in hypertension. Hypertriglyceridemia was linked to the rs2393791_T (1.14 (1.02–1.27);P=0.018), rs7310409_G (1.12 (1.01–1.25);P=0.031), rs1169310_G (1.15 (1.04–1.28);P=0.010), and rs1169313_CT+TT (1.24 (1.06–1.45);P=0.008) and high low density lipoprotein-cholesterol levels were associated with rs2259820_T (1.23 (1.07–1.41);P=0.004), rs2464196_T (1.22 (1.06–1.39);P=0.004), and rs2259816_T (1.18 (1.02–1.36);P=0.023). A 7-mer haplotype CATATAC (χ2=7.50;P=0.0062), constructed from the studied SNPs, was associated with MI, and CATATA implicated in T2DM (χ2=3.94;P=0.047). Hypertriglyceridemia was linked to TGCGGG (χ2=4.26;P=0.039), and obesity to ACGGGT (χ2=5.04;P=0.025). Our results suggest that theHNF1ais a common susceptibility gene for MI, T2DM, hypertension, and dyslipidemia.

We reported in a previous study that β-adrenoceptor blockers inhibit the Mg2+-dependent ATP-hydrolytic function of Na+/K+-ATPase. To determine if this action is a result of binding of β-blockers to the receptor sites that bind the digitalis glycosides, we performed displacement binding assays of eight β-blockers with [3H]-ouabain (OUA) in guinea pig myocardial microsomal preparations. In the first series of experiments, 10-200 µM of the β-blockers were displaced with 250 nM OUA. In the second set of experiments, 10-500 nM of OUA was displaced using 200 µM of the β-blockers. The drugs showed concentration-dependent receptor occupancy at the different OUA levels. Propranolol (PPN), metoprolol (MTP), and sotalol (STL) showed the strongest binding; nadolol (NDL), indenolol (IDN), and atenolol (ATN) had intermediate binding; carazolol (CRZ) and celiprolol (CLP) had the weakest binding properties. The results suggest that β-blockers may compete for the same binding sites with ouabain in their inhibition of the Na+/K+-ATPase. These actions may contribute to the mechanism for some of their cardiac effects, especially their proarrhythmic and arrhythmogenic actions.Key words: β-adrenoceptor blockers, antiarrhythmic agents, arrhythmogenic effects, Na+/K+-ATPase, ouabain binding.

We evaluated the role of the MEF2A as a risk factor for coronary artery disease (CAD) in 1186 subjects with angiographically documented disease compared with 885 CAD-free individuals in the Saudi population. Screening the gene revealed exon 11 as the most polymorphic of all coding regions, harbouring several substitution polymorphisms and insertion/deletions (indels) at a locus containing an 11 CAG trinucleotide chain and a CCGCCGCCA sequence, which introduced frameshifts and premature stop codons at nt146637 and nt146647, nt146780 or nt146783. While these indels were not significantly associated with CAD, a causative relationship was established for rs1059759 G>C [1.21(1.02-1.43); p=0.029], and a borderline one for rs34851361 A>G [1.22(0.9-1.54); p=0.088]. Importantly, a haplotype 1A-2G-3G-4A-5C-6G-7G-8A constructed from the studied SNPs was also associated with CAD [6.39(0.93-43.75); p=0.0052]. These results identify MEF2A gene as a susceptibility gene for CAD.

Background The study was designed to evaluate the association of GATA4 gene polymorphism with coronary artery disease (CAD) and its metabolic risk factors, including dyslipidaemic disorders, obesity, type 2 diabetes and hypertension, following a preliminary study linking early onset of CAD in heterozygous familial hypercholesterolaemia to chromosome 8, which harbours the GATA4 gene. Results We first sequenced the whole GATA4 gene in 250 individuals to identify variants of interest and then investigated the association of 12 single-nucleotide polymorphisms (SNPs) with the disease traits using Taqman chemistry in 4,278 angiographed Saudi individuals. Of the studied SNPs, rs804280 (1.14 (1.03 to 1.27); p = 0.009) was associated with CAD (2,274 cases vs 2,004 controls), hypercholesterolaemia (1,590 vs 2,487) (1.61 (1.03–2.52); p = 0.037) and elevated low-density lipoprotein-cholesterol (hLDLC) (575 vs 3,404) (1.87 (1.10–3.15); p = 0.020). Additionally, rs3729855_T (1.52 (1.09–2.11; p = 0.013)) and rs17153743 (AG + GG) (2.30 (1.30–4.26); p = 0.005) were implicated in hypertension (3,312 vs 966), following adjustments for confounders. Furthermore, haplotypes CCCGTGCC (χ 2 = 4.71; p = 0.041) and GACCCGTG (χ 2 = 3.84; p = 0.050) constructed from the SNPs were associated with CAD and ACCCACGC (χ 2 = 6.58; p = 0.010) with myocardial infarction, while hypercholesterolaemia (χ 2 = 3.86; p = 0.050) and hLDLC (χ 2 = 4.94; p = 0.026) shared the AACCCATGT, and AACCCATGTC was associated with hLDLC (χ 2 = 4.83; p = 0.028). A 10-mer GACCCGCGCC (χ 2 = 7.59; p = 0.006) was associated with obesity (1,631 vs 2,362), and the GACACACCC (χ 2 = 4.05; p = 0.044) was implicated in type 2 diabetes mellitus 2,378 vs 1,900). Conclusion Our study implicates GATA4 in CAD and its metabolic risk traits. The finding also points to the possible involvement of yet undefined entities related to GATA4 transcription activity or gene regulatory pathways in events leading to these cardiovascular disorders.

Objective.—To determine the relevance of angiotensin I–converting enzyme (ACE) gene polymorphism for coronary artery disease (CAD) in the Saudi population. Methods and Results.—DNA of 84 male Saudi patients with established CAD, 36 male controls who underwent angiography, and 327 healthy Saudi male blood donors was amplified by polymerase chain reaction, using oligonucleotide primers flanking the insertion (I)/deletion (D) sites in the polymorphic region of intron 16 of the ACE gene. Polymerase chain reaction amplification resulted in 490-bp (II), 190-bp (DD), or 490- and 190-bp (ID) fragments. The genotype II distribution was 16.7% in the control group, 7.3% in the blood donor group, and 7.2% in the patients with CAD, and the distribution for DD was 58.3%, 47.1%, and 41.0%, respectively. Notably, 61.9% (P < .0001) of CAD patients presented with angina on admission, and 52.4% had diabetes mellitus. Conclusions.—The results show no increased risk of CAD in association with either the II or DD genotypes in the Saudi population. However, further investigation of genotype II as a predictor for atherosclerosis rather than increased risk of coronary heart disease may be indicated.

The notion that the inhibition of the Mg2+-dependent ATP-hydrolytic function of the myocardial Na+-K+ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 µM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 µM, 25-44% at 100 µM, and 37-56% at 200 µM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 µM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+-K+ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.Key words: class I antiarrhythmic agents, proarrhythmias, Na+-K+ATPase, ouabain binding.

Background: The aim of this study was to look into the association, if any, of apoprotein-CIII variant allele with hypertriglyceridemia, hypercholesterolemia and coronary heart disease ( CHD). Patients and Methods: The prevalence of a C to G substitution in the 3' untranslated region of apoprotein-CIII was studied in a sample of 92 angiographed Saudi subjects, consisting of 65 males and 27 females. The subjects were genotyped by amplification followed by digestion of the gene fragment containing the polymorphic site with Sac I restriction enzyme. Results: The variant allele of apoprotein-CIII was found to be associated neither with hypertriglyceridemia nor with hypercholesterolemia. However, a significant association of this allele ( P

The notion that the inhibition of the Mg2+ -dependent ATP-hydrolytic function of the myocardial Na+ -K+ ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 microM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 microM, 25-44% at 100 microM, and 37-56% at 200 microM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 microM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+ -K+ ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.

1. The influence of amiodarone on [ 3 H]ouabain (OUA) binding to myocardial Na + , K + -ATPase was studied at various KCl concentrations in guinea pig heart microsomal preparations to test the hypothesis that the drug acts on the same receptor sites as cardiac glycosides. 2. First, a series of assays for OUA binding to Na + , K + -ATPase were performed in the range of 64–800 nM at 2.5, 5.0 and 10.0 mM K + concentration. The drug exhibited increasing binding tendency with increasing concentrations and elevation in K + levels. 3. Competitive binding assays were then performed at 256, 512 and 800 nM OUA in the presence of 50, 100 and 200 μM amiodarone at 5.0 mM KCl, respectively. At each OUA concentration, a concentration-dependent left-to-right shift was observed in the binding affinity with increasing amiodarone concentration. Similar assays at 2.5 and 10.0 mM K + showed the same trends. These effects were significant for 200 mM amiodarone at all K + levels and OUA concentrations. 4. Furthermore, different OUA concentrations were also shown to displace amiodarone in a concentration-dependent fashion. 5. These results indicate that amiodarone competes with OUA for specific binding sites on myocardial microsomal Na + , K + -ATPase. They lead to the conclusion that myocardial Na + , K + -ATPase is a possible receptor for some of the cardiac actions of amiodarone, such as its proarrhythmic effects.

Background The muscle Ras (MRAS) gene resides on chromosome 3q22.3 and encodes a member of the membrane-associated Ras small GTPase proteins, which function as signal transducers in multiple processes including cell growth and differentiation. Its role in cardiovascular disease is not fully understood yet. In a preliminary study in heterozygous familial hypercholesterolaemia, we identified a locus linking the early onset of coronary artery disease (CAD) to chromosome 3q.22 and elected to sequence the MRAS gene using the MegaBACE DNA analysis system. In the present study, we investigated the association of seven single-nucleotide polymorphisms (SNPs) at this locus with CAD and its dyslipidaemia-related risk traits in 4,650 Saudi angiographed individuals using TaqMan assays by the Applied Biosystems real-time Prism 7900HT Sequence Detection System. Results Among the studied SNPs, rs6782181 (p = 0.017) and rs9818870T (p = 0.009) were associated with CAD following adjustment for sex, age and other confounding risk factors. The rs6782181_GG also conferred risk for obesity (1,764 cases vs. 2,586 controls) [1.16(1.03–1.30); p = 0.017], hypercholesterolaemia (1,686 vs. 2,744) [1.23(1.02–1.47); p = 0.019], hypertriglyceridaemia (1,155 vs. 3,496) [1.29(1.01–1.45); p = 0.043] and low high-density lipoprotein-cholesterol (lHDL-chol) levels (1,935 vs. 2,401) [1.15(1.02–1.30); p = 0.023] after adjustment. Additionally, rs253662_(CT+TT) [1.16(1.01–1.32); p = 0.030] was associated with lHDL-chol levels. Interestingly, rs253662 (p = 0.014) and rs6782181 (p = 0.019) were protective against acquiring high low-density lipoprotein-cholesterol (hLDL-chol) levels (p = 0.014), while rs1720819 showed similar effects against CAD (p < 0.0001). More importantly, a 7-mer haplotype, ACCTGAC (χ 2 = 7.66; p = 0.0056), constructed from the studied SNPs, its 6-mer derivative CCTGAC (χ 2 = 6.90; p = 0.0086) and several other shorter derivatives conferred risk for obesity. hLDL-chol was weakly linked to CTAA (χ 2 = 3.79; p = 0.052) and CCT (χ 2 = 4.32; p = 0.038), while several other haplotypes were protective against both obesity and hLDL-chol level. Conclusion Our results demonstrate that the genomic locus for the MRAS gene confers risk for CAD, obesity and dyslipidaemia and point to the possible involvement of other genes or regulatory elements at this locus, rather than changes in the M-Ras protein function, in these events.